Use of the cathelicidin LL-37 and derivatives thereof for wound healing

ABSTRACT

Use of the antimicrobial cathelicidin peptide II-37, N-terminal fragments of LL-37 or extended sequences of LL-37 having 1-3 amino acids in the C-terminal end, for stimulating proliferation of epithelial and stromal cells and thereby healing of wounds, such as chronic ulcers. The cytotoxic effect of LL-37 may be reduced by including a bilayer-forming polar lipid, especially a digalactosyldiacylglycerol, in pharmaceutical compositions and growth media comprising LL-37.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 13/938,120filed Jul. 9, 2013, which is a divisional of co-pending application Ser.No. 13/193,131 filed on Jul. 28, 2011, now U.S. Pat. No. 8,506,994,which is a divisional of to application Ser. No. 12/232,215, filed onSep. 12, 2008, now U.S. Pat. No. 8,012,933 B2, which issued on Sep. 6,2011. Application Ser. No. 12/232,215 is a divisional of applicationSer. No. 10/543,659 filed on Jul. 28, 2005, now U.S. Pat. No. 7,452,864,which issued on Nov. 18, 2008. Application Ser. No. 10/543,659 is thenational stage of PCT International Application No. PCT/SE2004/000111filed Jan. 28, 2004, which claims the benefit of priority of ApplicationNo. SE 0300207-8 filed in Sweden on Jan. 29, 2003 and U.S. ApplicationNo. 60/444,964 filed on Feb. 5, 2003. The entire contents of each ofthese applications are hereby incorporated by reference.

TECHNICAL FIELD

The present invention refers to the peptide LL-37 and N-terminalfragments, as well as functional derivatives thereof, which peptides canbe used for cell proliferation, epithelial repair, and wound healing,and to a pharmaceutical composition comprising one or more of saidpeptides.

BACKGROUND OF THE INVENTION

Epithelia constitute the primary barrier between host and thepotentially harmful environment, and therefore the protection of thisinterface is vital. A wound represents a broken barrier and immediatelysets in motion a series of tightly orchestrated events with the purposeto promptly reinstate the integrity of the barrier. Urgent wound closurehas evolved in higher organisms, diverging from the time-consumingprocess of complete regeneration of tissue seen in lower species.Impaired wound healing represents a major challenge in clinical medicineranging from the relative delay in “normal” healing seen with increasingage to pathologic non-healing ulcers.

Chronic ulcers constitute a major clinical problem and although ourunderstanding of the physiologic wound process has increased over thepast decades only minor

Chronic ulcers constitute a major clinical problem and although ourunderstanding of the physiologic wound process has increased over thepast decades only minor therapeutic improvements have been attained.Distinct etiologies may underlie the development of ulcerations indifferent clinical conditions but, whatever the cause, non-healingulcers are characterized by an inability of the epithelium to migrate,proliferate and close the barrier defect. The most common type ofchronic skin ulcers is leg ulcers due to venous insufficiency. Thesepatients develop peripheral venous oedema with subsequent ulceration ofthe skin, whereas the arterial circulation is intact. Leg and footulcers due to arteriosclerotic deficiencies are less common.

In addition, skin ulcers develop in association with immune diseasessuch as pyoderma gangrenosum and vasculitis. Current treatment includeslong-term systemic immunosuppression and is not always effective.Epithelial defects and ulcers in the oral, genital and gastrointestinalmucous membranes are common and cause much distress. The underlyingpathomechanisms are not always clear, such as in aphtae and erosivelichen and treatment is poor.

Traditional wound care involves removal, mechanically or enzymatically,of necrotic debris to allow formation of granulation tissue. Wounds thatare heavily colonized with bacteria may require antiseptic treatment toprevent invasive infection. Numerous topical anti-microbial agents areused, such as iodine, chlorhexidine, hydrogen peroxide, silver andantibiotics, but the risk of toxic effects of these agents on the matrixand the neoepidermis must be considered. Once the wound is clean ofnecrotic tissue, dressings should be used to promote granulation tissueformation. A large variety of such dressings are available and numerousanimal studies and clinical trials have demonstrated their beneficialeffect on wound healing.

A certain proportion of wounds remain therapy-resistant and there isneed for additional treatment. During the past decade there has beenmuch focus on the potential use of growth factors to accelerate woundrepair. Growth factors are molecules, which control cellular processesthat are critical in tissue repair, including cell migration,proliferation, angiogenesis and de novo synthesis of extracellularmatrix. The beneficial effect of such growth factors has been suggestedin a wide variety of trials (Scharffetter-Kochanek et al., Basic ResCardiol 93:1-3, 1999). However, to date growth factor treatment ofchronic ulcers has been largely disappointing in clinical practice. Atpresent becaplermin (Regranex®), licensed in U.S. and Europe but not inSweden, is the only growth factor for use, preferentially in diabeticfoot ulcers. The reasons for clinical failure of growth factors in thetreatment of chronic ulcers are thought to involve delivery problems andrapid degradation.

In parallel, there has been development of tissue therapies usingauto-logous and allogenic materials in bioengineered human skinequivalents. Cultured epidermal keratinocytes constitute a functioningtreatment for coverage of large areas of injured skin in e.g. burnpatients, but is expensive, time consuming and requires laboratoryfacilities. To provide a dermal substrate multiple strategies have beenused such as acellular human cadaver and bovine collagen with or withoutcells. All methods available have considerable disadvantages such aspotential transmission of disease and high costs and are hardly suitedfor basic wound care.

Antimicrobial peptides are effector molecules of the innate immunesystem, which serve to protect the host against potentially harmfulmicroorganisms. They are conserved through evolution and are widespreadin nature. In human, only a handful has been identified so far; amongwhich the defensins and the human cathelicidin antimicrobial peptidehCAP18 have been implicated in epithelial defense (Selsted et al., JBiol Chem 258:14485-14489, 1983).

WO 96/08508 relates to the human polypeptide FALL-39, as well as topharmaceutical compositions containing said peptide and having anantimicrobial activity against bacteria. The peptide was named FALL-39after the first four amino acid residues and consisted of the 39 aminoacid C-terminal part of a proprotein concomitantly identified by threeseparate groups (Cowland et al., FEBS, 1995; Agerberth et al., Proc NatlAcad Sci USA 1995; Larrick et al., FEBS Letters 1996). The peptide wasshown to have potent antimicrobial activity against both gram-positiveand gram-negative bacteria. Further characterization of the C-terminalpeptide demonstrated a shorter sequence comprising 37 amino acidsexcluding the first two (FA) resulting in LL-37, which is the acceptedcurrent designation (Gudmundsson et al., Eur J Biochem 238:325-332,1996).

The proprotein was named hCAP18, human cationic anti-microbial protein,and is a member of the cathelicidin family of proteins consisting ofcathelin, which has been conserved through evolution and a C-terminalpart, variable in different species. In man, hCAP18 is the only memberof this protein family, whereas in other species, such as mouse and pig,there are several members. The C-terminal peptide LL-37 is thought tofunction extracellularly and there is no evidence for intracellularcleavage of the proprotein. hCAP18/LL-37 is present in leukocytes and inbarrier organs such as skin, mucous membranes, respiratory epitheliumand reproductive organs. The localization of hCAP18/LL-37 to barrierepithelia seems to be consistent with a protective role for the peptidein preventing local infection and systemic microbial invasion. LL-37 isdescribed as a cysteine-free peptide that can adopt an amphiphatic, orin other words amphiphilic, α-helical conformation. A high cationicityin combination with a stabilized amphiphatic α-helical structure seemsto be required for the anti-microbial effect of such peptides againstgram-positive bacteria and fungi, as has been shown experimentally(Gianga-spero et al., Eur J Biochem 268:5589-5600, 2001). Theamphiphatic and α-helical structure seems to be less critical forkilling of gram-negative bacteria. In association with inflammationhCAP18/LL-37 is upregulated in skin epithelium (Frohm et al., J BiolChem 272:15258-15263, 1997) and mucous membranes (Frohm Nilsson et al.,Infect Immun 67:2561-2566, 1999).

PRIOR ART

Dorschner et al., J Invest Dermatol 117:91-97, 2001, demonstrated thatthe expression of cathelicidins was increased in human and murine skinafter incision, and that lack of the murine homologue cathelicidin genefails to protect against invasion of Group A streptococci in such mice.

WO 96/09322, Children's Medical Center Corporation, discloses that theantibacterial peptide PR-39 possesses syndecan-1 and -4 inductiveactivity and therefore simultaneously could reduce infection and, as asynducin, influence the action of growth factors, matrix components, andother cellular effectors involved in tissue repair. The synducins couldbe administered in a pharmaceutical carrier, such as conventionalliposomes.

EP 0 935 965 A1, Toray Industries, Inc., refers to an antipylori agentcontaining an anti-microbial peptide, such as the porcine peptide PR-39,as an active agent. It is concluded that exogeneous administration ofPR-39 has anti-microbial activity against Helicobacter pylori andaccelerates healing of gastric ulcers in rat. FALL39 is mentioned as oneof the members of the cathelin family.

U.S. Pat. No. 6,255,282, Helix Biomedix, Inc., discloses novel syntheticlytic peptides sharing structural and functional properties of differentknown lytic peptides. Especially a peptide of 18 to about 40 amino acidsand having an α-helical conformation is described. The lyticcathelicidin peptides, however, are not mentioned.

Frohm Nilsson, Thesis, Karolinska Institutet, Stockholm 2001,concomitantly demonstrated that human cathelicidin anti-microbialprotein, hCAP 18, is induced in human skin wounding, with high levelsand release of active C-terminal peptide, LL-37, in physiologicalhealing but not in chronic non-healing ulcers. hCAP18 was detected inthe wound bed and in the epithelium during normal wound healing but wasabsent in the epithelium of chronic leg ulcers and was detected only inthe wound bed and stroma. It was speculated that low levels of hCAP18and the lack thereof in the epithelium of chronic ulcers contribute toimpaired healing.

Zasloff, Nature 415:389-395, 2002, in a review of anti-microbialpeptides discusses the diverse applications, which have beendemonstrated for said peptides as anti-infective agents, andanti-microbial peptides in pharmaceutical development are described.

EP 1 358 888 A1, Bals et al., having a date of publication of Nov. 5,2003, relates to the use of the peptide LL-37 for prevention ortreatment of a disease caused by reduced blood flow and arteriosclerosisand for treatment of wounds due to reduced arterial blood supply. Theability of LL-37 to induce formation of new blood vessels and tostimulate proliferation of endothelial cells is shown. The inventionrelates entirely to the angiogenetic effect and there is no mentioningof epithelia.

Although a therapeutic use of anti-microbial peptides, in particularLL-37, has been suggested, this has so far not been realized. At highconcentrations of the peptide, LL-37 exerts a cytotoxic effect. Thepotential cytotoxic effects exerted by LL-37 are, however, inhibited inthe presence of serum, but pharmaceutical formulations containing serumshould be avoided due to risk for transmitting diseases, restrictedaccessibility and high costs.

SUMMARY OF THE INVENTION

The human anti-microbial peptide hCAP18 is up-regulated in skinepithelium as a normal response to injury. However, in chronicnon-healing leg ulcers only low levels of hCAP18 were found. Notably, inthe chronic leg ulcers, hCAP18 and LL-37 were entirely absent in theepithelium but present in the inflammatory infiltrate in the wound bedand in the stroma. We have now shown that hCAP18 is induced duringre-epithelialization of organ-cultured skin wounds, and that thisre-epithelialization was inhibited by antibodies against LL-37 in aconcentration-dependant manner. These findings suggest that LL-37 playsa crucial role in wound closure, functioning as a growth factor. Theinvention concerns the use of LL-37 or a new synthetic peptide derivedfrom LL-37 or a functional derivative thereof, to compensate for thelack of natural LL-37 produced in vivo.

It was also shown that up-regulation of hCAP18 and/or adding LL-37peptide stimulate proliferation of normal epithelial and stromal cells,suggesting that normal wound healing and epithelial regeneration couldalso be enhanced.

It was also found that the cytotoxicity of LL-37 could be reduced in acomposition comprising certain lipids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of the 18 kDa hCAP18 protein consisting ofa signal peptide, S.P., the conserved cathelin part, and theanti-microbial peptide LL-37, which is enzymatically cut off in vivo.

FIG. 2 is a schematic drawing of the cathelicidin protein family,illustrating the diversity of C-terminal peptides in different species.

FIGS. 3A, 3B, and 3C show the cDNA sequence (SEQ ID NO: 21) of thepIRES2-EGFP vector including the coding sequence for hCAP18, used fortransgenic expression of hCAP 18.

DESCRIPTION OF THE INVENTION

The present invention refers to a peptide having a sequence of at least20 amino acids of the N-terminal fragment of LL-37, with the provisothat LL-37 is excluded, as well as to pharmaceutically acceptable saltsand derivatives thereof. LL-37 has the amino acid sequence SEQ ID NO 1:

-   -   H-Leu-Leu-Gly-Asp-Phe-Phe-Arg-Lys-Ser-Lys-Glu-Lys-Ile-Gly-Lys-Glu-Phe-Lys-Arg-Ile-Val-Gln-Arg-Ile-Lys-Asp-Phe-Leu-Arg-Asn-Leu-Val-Pro-Arg-Thr-Glu-Ser-OH.

The N-terminal sequence of LL-37 refers to a sequence beginning with theamino acid residue number 1 of leucine, Leu.

Pharmaceutically acceptable salts contain for instance the counterionsacetate, carbonate, phosphate, sulphate, trifluoroacetate, and chloride.A preferred salt is the acetate. Esters and amides are examples ofpharmaceutically acceptable derivatives.

The peptide of the invention should have an amino acid chain of no morethat 40 amino acids. The invention refers to a peptide having thesequence of LL-37 to which 1-3 amino acids have been added in theC-terminal end. Any amino acid selected from Ala, Arg, Asn, Asp, Cys,Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr,Val, as well as derivatives thereof can be added. An example of apeptide having 38 amino acids, LL-38, SEQ ID NO 19, has the sequence ofLL-37 to which serine has been added in the C-terminal end.

The invention especially refers to a peptide having a sequence of atleast 20 amino acids and selected from the group consisting of LL-36,LL-35, LL-34, LL-33, LL-32, LL-31, LL-30, LL-29, LL-28, LL-27, LL-26,LL-25, LL-24, LL-23, LL-22, LL-21 and LL-20, having the sequence SEQ IDNO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7,SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, andSEQ ID NO 18, respectively.

Preferred peptides are selected from the groups consisting of LL-36,LL-35, LL-34, LL-33, LL-32, LL-31, LL-30, LL-29, LL-28, LL-27, LL-26,and LL-25.

The amino acid sequences of the peptides of the invention are given inthe following table.

SEQ ID Pep- NO tide Amino Acid Sequence 1 LL-LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES 37 2 LL-LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTE 36 3 LL-LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRT 35 4 LL-LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPR 34 5 LL-LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVP 33 6 LL-LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLV 32 7 LL-LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNL 31 8 LL- LLGDFFRKSKEKIGKEFKRIVQRIKDFLRN30 9 LL- LLGDFFRKSKEKIGKEFKRIVQRIKDFLR 29 10 LL-LLGDFFRKSKEKIGKEFKRIVQRIKDFL 28 11 LL- LLGDFFRKSKEKIGKEFKRIVQRIKDF 27 12LL- LLGDFFRKSKEKIGKEFKRIVQRIKD 26 13 LL- LLGDFFRKSKEKIGKEFKRIVQRIK 25 14LL- LLGDFFRKSKEKIGKEFKRIVQRI 24 15 LL- LLGDFFRKSKEKIGKEFKRIVQR 23 16 LL-LLGDFFRKSKEKIGKEFKRIVQ 22 17 LL- LLGDFFRKSKEKIGKEFKRIV 21 18 LL-LLGDFFRKSKEKIGKEFKRI 20 19 LL- LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTESS 38

The new peptides can be used as a medicament for cell proliferation,epithelial regeneration, healing of normal or chronic wounds, and asantimicrobial agents.

The new peptides are believed to have the potential to form an α-helicalstructure at physiological conditions.

According to another aspect the invention refers to the use of a peptidehaving an amino acid sequence selected from the group consisting of

-   -   a) SEQ ID NO 1;    -   b) a sequence containing at least 20 amino acids of the        N-terminal fragment of SEQ ID NO 1;        and pharmaceutically acceptable salts or derivatives thereof,        which peptide enhances proliferation of epithelial and/or        stromal cells through a non-lytic mechanism, for the preparation        of a medicament for epithelial regeneration, and healing of        wound epithelium and stroma.

The present invention especially refers to the use of the peptide LL-37having the amino acid sequence SEQ ID NO 1, in the form of a salt,preferably an acetate salt.

The invention also refers to the use of a peptide, selected from thegroup consisting of LL-20 to LL-36, as stated above.

LL-37, as well as LL-25 to LL-36, possesses a net positive charge(+5-+7) at neutral pH due to the cationic amino acid residues of lysineand arginine in the primary structure. Especially LL-34 and LL-35 havenet positive charge of 7. The other amino acid residues arenonpolar/hydrophobic or polar and neutral, or, to a less extent, polarand negatively charged, which makes the whole peptide moleculeamphiphatic. Peptides of this type interact electrostatically with thenegatively charged phospholipid microbial cell walls inserting thehydrophobic face into the bilayer. A reduction of either hydrophobicityand/or charge reduces the anti-microbial effect of the peptides. Thecytotoxic effect exerted by the peptides against host cells, oftenassessed as hemolytic activity, is shown to correlate with theiranti-microbial effects (Chen et al., FEBS Lett 236:462-466, 1988).Various studies have confirmed that this is true also for otheramphiphatic α-helical anti-microbial peptides.

Studies of the C-terminal peptide, having a length of 37 amino acids, ofrabbit CAP18 (Cap18₁₀₆₋₁₄₂) show that broad-spectrum antibacterialactivity is retained in the highly basic 20 residue N-terminal sequence,but not if the N-terminus is truncated (Larrick et al., AntimicrobAgents Chemother 37:2534-2539, 1993).

LL-37, as well as the new peptides LL-20 to LL-36, can be synthesizedusing an automatic peptide synthesizer and standard methods for peptidesyntheses.

The invention especially refers to the use of the LL-37 peptide oranyone of the peptides LL-20 to LL-36 for the preparation of amedicament for treatment of chronic ulcers. Said chronic ulcers can bedue to venous insufficiency, such as leg ulcers, metabolic dysfunction,such as diabetes, or immunological diseases, such as vasculites, andpyoderma gangrenosum. The peptides of the invention can also be used fortreatment of wounds due to trauma or burns. The described peptides canespecially be used for regeneration of epithelial tissue, and to enhanceepidermal regeneration following microdermabrasion.

In addition to being toxic to the cell, LL-37 is rapidly degraded in thewound environment. Serine proteinase 3 was recently shown to beresponsible for extracellular cleavage of hCAP18 (Sorensen et al., Blood97:3951-3959, 2001).

In order to prevent decomposition of the peptide and also for reducingthe intrinsic cytotoxicity, the peptide can be formulated with a polarlipid carrier. Said formulation should facilitate the administration ofthe peptide to the wound and will in addition provide a sustainedrelease of the peptide after administration. The stability of thepeptide will be improved both in vivo and in vitro.

Another object of the invention is thus a pharmaceutical compositioncomprising an anti-microbial cathelicidin peptide in the form ofpharmaceutically acceptable salts or derivatives thereof in combinationwith a carrier consisting of a bilayer-forming polar lipid and anaqueous solution.

The cathelicidin peptide can, in addition to LL-37 in human, be derivedfrom different animal species, and is for example SC5 from sheep, Bac5from cow, PR-39 from pig, CRAMP from mouse, and p15 from rabbit, seeFIG. 2.

A bilayer normally refers to the lamellar arrangements of polar lipidsin water. The acyl chains form the internal hydrophobic part and thepolar head-groups the hydrophilic part of the bilayer. As examples ofsuch polar bilayer-forming lipids, either of natural or syntheticorigin, can be mentioned phosphatidylcholine, phosphatidylglycerol,digalactosyldiacylglycerol, sphingomyelin and the like. Depending on theconcentration of said polar lipids in polar solvents, such as water,liposomes or viscous gels of the lamellar liquid crystalline type may beformed.

The pharmaceutical composition especially comprises a peptide having anamino acid sequence selected from the group consisting of

-   -   a) SEQ ID NO 1;    -   b) a sequence containing at least 20 amino acids of the        N-terminal fragment of SEQ ID NO 1;        in the form of pharmaceutically acceptable salts or derivatives        thereof in combination with a carrier consisting of a        bilayer-forming polar lipid and an aqueous solution.

Preferred bilayer-forming polar lipids to be mixed or formulated withthe peptide are those, which are neutral in charge. Especially usefulare the digalactosyldiacylglycerols, and other glycolipids, such as theglycosyl ceramides, either natural or synthetic, in which a non-ioniccarbohydrate moiety constitutes the polar head-group. Less preferred,but still useful, are those polar lipids, which are zwitterionic andneutral at physiological conditions, such as phosphatidylcholine,phosphatidylethanolamine, and sphingomyelin. Least preferred are thosepolar lipids, which are negatively charged and thus form strongcomplexes with the positively charged peptide.

According to the invention said bilayer-forming polar lipid carrier ispreferably selected from the group consisting of phospholipids,galactolipids and sphingolipids.

An especially preferred bilayer-forming polar lipid isdigalactosyldiacylglycerol or polar lipid mixtures rich indigalactosyldiacylglycerols due to the extremely good cutaneoustolerability of this class of polar lipids. Digalactosyldiacylglycerolis a class of lipids belonging to the glycolipid family, well knownconstituents of plant cell membranes. One of the most abundant classescontains two galactose units, and the commonly used nomenclature andabbreviation of this is digalactosyldiacylglycerol, DGDG, sometimesreferred to as galactolipids. Galactolipids, primarily DGDG andDGDG-rich materials have been investigated and found to be surfaceactive material of interest in industrial applications such as food,cosmetics, and pharmaceutical products. WO 95/20944 describes the use ofDGDG-rich material, a “galactolipid material”, as a bilayer-formingmaterial in polar solvents for pharmaceutical, nutritional and cosmeticuse. Said application does not disclose the use of galactolipids incombination with peptides and proteins in general, particularly not apeptide of the present invention.

According to a preferred aspect the invention refers to a pharmaceuticalcomposition wherein the bilayer-forming polar lipid carrier is a polarlipid mixture rich in digalactosyldiacylglycerols.

Another preferred aspect of the invention is a pharmaceuticalcomposition wherein the peptide is in the form of acetate. A preferredpeptide is LL-37 in the form of an acetate salt. Especially preferred isa pharmaceutical composition comprising a combination of an acetate ofLL-37 and CPL-Galactolipid as the bilayer-forming lipid carrier.CPL-Galactolipid is a trademark for a galactolipid fraction consistingof 50-70% by weight of digalactosyldiacylglycerols and 30-50% or otherpolar lipids.

The ratio between the peptide in the form of a salt and a galactolipidcarrier in the pharmaceutical composition should preferably be 1:5 to1:50, especially 1:10-1:25 by weight.

In addition to the bilayer-forming lipid the carrier also contains anaqueous solution. An aqueous solution refers to a solution havingphysiologically or pharmaceutically acceptable properties regarding pH,ionic strength, isotonicity etc. As examples can be mentioned isotonicsolutions of water and other biocompatible solvents, aqueous solutions,such as saline and glucose solutions, and hydrogel-forming materials.The aqueous solution can be buffered, such as phosphate-buffered saline,PBS.

The pharmaceutical composition can in addition comprise pharmaceuticallyacceptable excipients, such as a preservative to prevent microbialgrowth in the composition, antioxidants, isotonicity agents, colouringagents and the like. In aqueous suspensions the compositions can becombined with suspending and stabilising agents.

The colloidal nature of the composition makes it possible to prepare thecomposition aseptically by using a final sterile filtration step.

In order to form a gel the peptide can be preferably formulated with ahydrogel-forming material. Examples of hydrogel-forming materials aresynthetic polymers, such as polyvinylalcohol, polyvinylpyrolidone,polyacrylic acid, polyethylene glycol, poloxamer block copolymers andthe like; semi-synthetic polymers, such as cellulose ethers, includingcarboxymethylcellulose, hydroxyethylcellulose, hydroxy-propylcellulose,methylcellulose, methylhydroxypropylcellulose andethylhydroxy-ethylcellulose, and the like; natural gums, such as acacia,carragenan, chitosan, pectin, starch, xanthan gum and the like.

It is advantageous to use a hydrogel which is muco-adhesive. In thatrespect it is particularly useful to use hyaluronic acid and derivativesthereof, cross-linked polyacrylic acids of the carbomer andpolycarbophil types, polymers that readily form gels, which are known toadhere strongly to mucous membranes.

It is also advantageous to use block copolymers of the poloxamer type,i.e. polymers consisting of polyethylene glycol and polypropylene glycolblocks. Certain poloxamers dispersed in water are thermoreversible: atroom temperature they are low viscous but exhibit a marked viscosityincrease at elevated temperatures, resulting in a gel formation at bodytemperature. Thereby the contact time of a pharmaceutical formulationadministered to the relatively warm wound may be prolonged and thus theefficacy of the incorporated peptide may be improved.

The pharmaceutical composition of the invention can be formulated fortopical or enteral, that is oral, buccal, sublingual, mucosal, nasal,bronchial, rectal, and vaginal administration.

Non-limiting examples of pharmaceutical compositions for topicaladministration are solutions, sprays, suspensions, emulsions, gels, andmembranes. If desired, a bandage or a band aid or plaster can be used,to which the pharmaceutical composition has been added. Tablets,capsules, solutions or suspensions can be used for enteraladministration.

According to another aspect the invention refers to the use of a peptidehaving an amino acid sequence selected from the group consisting of

-   -   a) SEQ ID NO 1;    -   b) a sequence containing at least 20 amino acids of the        N-terminal fragment of SEQ ID NO 1;        in the form of pharmaceutically acceptable salts or derivatives        thereof for proliferation of epithelial and/or stromal cells in        vitro through a non-lytic mechanism.

Said proliferation can especially be used for proliferation of humanautologous epithelial and stromal cells in vitro.

The invention also refers to a growth medium for culturing eukaryoticcells, such as epithelial and/or stromal cells, which comprises LL-37 ora peptide as described in combination with a basal medium. Acytotoxicity reducing agent can be added, such as serum. ApolipoproteinA-I (apoA-I) has been found to be the main LL-37 binding protein inhuman plasma and works as a scavenger of LL-37 (Wang et al, J Biol Chem273:33115-33118, 1998; Sorensen et al, J Biol Chem 274:22445-22451,1999), suggesting a mechanism involved in the regulation of acathelicidin peptide. The cytotoxicity reducing agent can also be abilayer-forming polar lipid, such as a lipid selected from the groupconsisting of phospholipids, galactolipids and sphingolipids, asdescribed above.

The basal medium of the growth medium of the invention is based ondouble-distilled water, and a number of the following ingredients:inorganic salts, phenol red, glucose, thymidine, hypoxanthinine, HEPES,sodium pyruvate, aminopterin, amino acids and vitamins. For culturing ofepithelial cells, such as e.g. keratinocytes, in vitro the growth mediumcan consist of basal medium and a growth promoting kit including a)LL-37 peptide in a salt solution, b) penicillin+streptomycin, c)insulin, d) transferrin, e) triiodotyronine, f) hydrocortisone, g)choleratoxin, and a selected cytotoxicity reducing agent, such as serumor a polar lipid. For culturing of stromal cells, such as e.g.fibro-blasts, in vitro, a growth medium can consist of basal medium anda growth promoting kit including a) LL-37 peptide in a salt solution, b)penicillin+streptomycin, and a selected cytotoxicity reducing agent,such as serum or a polar lipid.

Another object of the invention is a method of enhancing the expansionof human autologous epithelial and stromal cells in vitro for celltransplantation in vivo, wherein cells are isolated from an excisedpiece of healthy skin, said isolated cells are cultivated in vitro in agrowth medium according to the invention, and the cultivated cells aresubsequently harvested and used for treatment of wounds, such as burninjuries and ulcers.

The invention also refers to a growth promoting kit comprising the LL-37peptide or a peptide as described, and a cytotoxicity reducingbilayer-forming polar lipid, optionally in combination with antibiotics,basal media, and other conventional additives in separate containers.

According to still another aspect the invention refers to transfectionof a full-length hCAP 18 cDNA construct into autologous humankeratinocytes for cell transplantation of ulcers and burns. The cDNAconstruct is designed to allow regulation of hCAP18 gene expression by aswitch mechanism (Resnitzky et al., Mol Cell Biol 14:1669-1679, 1994).Autologous human keratinocytes are obtained from a healthy skin pieceexcised from the patient. The keratinocytes are isolated and expanded incell culture as described. The cDNA construct is transfected intokeratinocytes. The transfected keratinocytes are further expanded invitro and given back to the patient.

The invention especially refers to the use of a gene constructcomprising the complete cDNA sequence of hCAP18 having the sequence SEQID NO 20 for transfection of epithelial and/or stromal cells in order toenhance proliferation of said cells.

EXAMPLES Example 1 Preparation of Synthetic Peptides

The LL-37 peptide was synthesized according to solid phase synthesiswith the 9-fluorenylmethoxycarbonyl/tert-butyl strategy. The crudepeptide, as the trifluoroacetate salt, was purified with HPLC andfinally isolated by lyophilization (lot 971/26, from PolyPeptideLaboratories A/S, HiHerod, Denmark). The purity was determined by meansof HPLC and area integration and was found to be 99%. The molecularweight was analyzed using mass spectrometry and corresponded to thetheoretical value of 4493 g/mol as the free base. Analysis ofcomposition of amino acids showed that the relative amounts of eachamino acid corresponded with the theoretical values for LL-37. Thepeptide content was calculated from the results from the amino acidanalysis and found to be 73%, the remainder being counterions andresidual solvent.

Several batches of LL-37 were synthesized, and the LL-37 peptide used inthe following Examples 2 and 5 was in the form of the acetate salt.

The peptides LL-36 and LL-38 were synthesized correspondingly, in theform of acetate.

The different peptides used in the following examples and tests were asfollows.

Purity Peptide content Manufact. Peptide Counterion Lot area-% % (w/w)Used in Year LL-37 Trifluoroacetate YS 5253 98 Ex. 3, 4, 6, 7 1997 Test3 LL-37 Trifluoroacetate 971/26 99 73 Test 5 2002 LL-37 Acetate 990/37/A99 83 Ex. 2, 5 2003 Test 4 LL-38 Acetate 990/38 Test 4 2003 LL-36Acetate 990/39 Test 4 2003

Example 2 Preparation of a Pharmaceutical Composition Comprising aMixture of LL-37 Peptide and Lipid Carrier

A pharmaceutical composition was prepared using the followingingredients:

Ingredient Concentration LL-37 100 ppm* CPL-Galactolipid 0.20% 2.6%Glycerol in sterile ad 100% water *ppm = parts per million (by weight)The peptide LL-37, as the acetate salt (lot 990/37/A), and the lipidcarrier, CPL-Galactolipid, obtained from Lipocore Holding AB, a lipidmaterial rich in digalactosyl-diacylglycerols and prepared from oats,were weighed in a 50 ml glass flask. The two ingredients were gentlymixed and then the glycerol solution was added. The mixture was shakenvigorously for 120 min and then allowed to stand for 1 h. The resultingcomposition was a fine, homogenous dispersion. It was kept refrigerateduntil use.

Example 3 Preparation of Aqueous Mixtures Comprising the LL-37 Peptideand a Lipid Carrier

Mixtures of LL-37, as the trifluoroacetate salt (lot 971/26) and apolar, bilayer-forming lipid carrier were prepared using the followingingredients (percentages in weight by weight):

TABLE 1 Ingredient A1 A2 B1 B2 C1 C2 LL-37 100 ppm — 90 — 92 —CPL-Galactolipid 0.19% 0.20% — — — — Epikuron 200 — — 0.19% 0.19% — —CPL-Sphingomyelin — — — — 0.19% 0.19% DMEM ad 100% ad 100% ad 100% ad100% ad 100% ad 100%

CPL-Galactolipid, obtained from Lipocore Holding AB, is achromatographically purified galactolipid fraction from oats, Epikuron200, obtained from Lucas Meyer GmbH, is phosphatidylcholine fromsoybean, and CPL-Sphingomyelin, obtained from Lipocore Holding AB, ischromatographically purified sphingomyelin from bovine milk. DMEM,Dulbecco's Modified Eagle Medium, from Invitrogen Corp. is an aqueoussolution containing inorganic salts, glucose, phenol red, amino acidsand vitamins

The peptide LL-37 and the lipid carrier were weighed in a glass flaskand then DMEM was added. The resulting dispersions were vigorouslyshaken, using a Heidolph Promax mixer at a frequency of 200/min for 1.5h, and allowed to equilibrate and settle for about 3 h at roomtemperature. A visual assessment was then made and the following resultswere obtained: All samples were turbid dispersions and there were nodifferences in turbidity between any of the samples B1, B2, C1, and C2.The only observed difference was between samples A1 and A2: the former,containing the peptide, was significantly less turbid than the latter,without the peptide. Sample A2 was slightly less turbid than, in turn,samples B1, B2, C1, and C2. These observations indicate a strongerinteraction between the two components in sample A1, which results in asmaller average particle size of the dispersion, compared to thepeptide-free sample A2, but also compared to the rest of thecorresponding samples. After one day of storage at room temperaturesamples A1 and A2 were unchanged, i.e. both were homogeneous dispersionsand A1 less turbid than A2, whereas the four other samples hadconsiderable sediments on the bottom of the glass flasks.

All three mixtures of peptide and polar lipid carrier are useful forvarious purposes, e.g. as delivery systems and for tests in cellcultures; however, since the shelf-life of the mixtures of peptide andgalactolipid is considerably longer (no sediment-ation) than that of theothers, said mixtures are the most preferred for practical use.

Example 4 Preparation of Aqueous Mixtures Comprising a Mixture of LL-37Peptide and Lipid Carrier

Samples of LL-37 as trifluoroacetate (lot 971/26) and a polar,bilayer-forming lipid carrier were prepared using the followingingredients (percentages in weight by weight):

TABLE 2 Ingredient Sample D Sample E Sample F Sample G Sample H Sample ISample J LL-37 96 ppm 100 ppm 100 ppm 103 ppm 100 ppm 100 ppm 100 ppmCPL-Galactolipid 0.21% — — — 0.20% — — PC from soybean, 40% — 0.21% — —— — — PC from egg yolk, 60% — — 0.21% — — — DOPC, 99% — — — 0.20% — — —PC from soybean, 70% — — — — — 0.20% — PC from soybean, 94% — — — — —0.20% PBS ad 100% ad 100% ad 100% ad 100% ad 100% ad 100% ad 100%

CPL-Galactolipid, manufactured by LTP Lipid Technologies Provider AB, isa chromatographically purified galactolipid fraction from oats. Thevarious phospholipids used were phosphatidylcholine (PC) from soybean,approximately 40% (Sigma; P-3644); PC from dried egg yolk, approximately60% (Sigma; P-5394); synthetic dioleylphosphatidylcholine (DOPC),approximately 99% (Sigma; P-6354); PC from soybean, approximately 70%(Lipoid S75); and PC from soybean, approximately 94% (Lipoid S100). PBSis phosphate-buffered saline from Invitrogen Corp. (Dulbecco's; cat. no.14190-094).

All the investigated polar lipids have chain melting phase transitiontemperatures well below 0° C., i.e., in the range of −10 to −15° C.,when fully hydrated.

The peptide LL-37 and the lipid carrier were weighed in a 100 ml glassflask and then PBS was added. The total volume was about 30 ml. Thesamples were vigorously shaken, using an ST mixer (type B1, E. Bilchler,Tübingen) set at 5.5 (corresponding to an approximate frequency of150/min) for 2 h, and allowed to equilibrate and settle for about 30 minat room temperature. The turbidity of the resulting dispersions was thenrecorded at 400-800 nm on a Shimadzu UV-VIS Spectrophoto-meter UV-160A.The measurements were made against pure water at room tempera-ture usinga 10 mm cuvette cell. Turbidity data in Table 3 are presented as %trans-mission at 600 nm. Visual assessments of the dispersions were alsomade. Turbidity measurements were repeated after one and two days ofstorage at room temperature of the dispersions.

TABLE 3 Turbidity Data Turbidity (λ = 600 nm) Sample D Sample E Sample FSample G Sample H Sample I Sample J 30 min 64.1% 70.9% 5.1% 1.7% 68.6%18.6% 1.4%  1 day 57.3% 65.6% — — 67.0% 19.8% —  2 days 57.2% 65.5% — —66.9% 20.5% —

From the visual assessments it was concluded that all mixtures formedmore or less turbid dispersions; samples D, E, H, and I formed the leastturbid dispersions, manifested in the highest transmission of light inTable 3, whereas samples F, G, and J formed the most turbid dispersionsand consequently gave rise to the lowest trans-mission of light detectedby the spectrophotometer. After one day of storage at room temperature,samples F, G, and J with the initially high turbidity (low transmission)had all sedimented and were not measured. Samples D, E, H, and I wereall stable dispersions and resulted in reproducible turbidity data,after one and two days after preparation.

Samples D and H are duplicates, both containing CPL-Galactolipid butsample H had a slightly higher weight ratio of peptide to galactolipid.This resulted in a slightly lower turbidity (higher transmission) insample H suggesting that the interaction between peptide and lipid inthis sample is stronger than that in sample D, leading to smallercomplexes/aggregates which give rise to lower turbidity.

Samples D, E, H, and I were further monitored with respect to colloidalstability at 2-8° C. for 2 months.

TABLE 4 Stability data Sample Appearance Stability D fine turbiddispersion, slight sedimentation, easy acceptable to redisperse sedimentE turbid dispersion, slight sedimentation; microbial not growthacceptable H fine turbid dispersion, slight sedimentation, easyacceptable to redisperse sediment I turbid dispersion, slightsedimentation; microbial not growth acceptable

These data and observations show that two mixtures of peptide and polarlipid carrier are better than the rest of the tested mixtures. Thecarriers containing CPL-Galactolipid (sample D and H) and PC fromsoybean, ca 40% (sample E) gave rise to the most finely dispersedsystems with the longest colloidal stability; however, it is onlyCPL-Galactolipid which is acceptable for pharmaceutical use, since thephospholipid material with only 40% phosphatidylcholine may be used fortechnical applications only. These data again demonstrate the usefulnessof the galactolipid material in various pharmaceutical applications,e.g. as a carrier system for peptides.

Example 5 Preparation of Aqueous Mixtures Comprising Varying Contents ofLL-37 Peptide and Varying Contents of Galactolipid

A stock solution of LL-37 peptide (acetate salt; lot 990/37/A) in PBS,995 ppm, and a stock solution of CPL-Galactolipid, 1.00%, in PBS wereprepared. Aliquots of the stock solutions plus additional PBS were mixedin 20 ml glass vials with rubber stoppers and aluminum caps. Thecompositions of the mixtures are presented in Table 5. Afterequilibration at room temperature for 1 h, the vials were shaken inhorizontal position on an ST mixer (type B1, E. Büehler, Tübingen), setat 7.5 (corresponding to an approximate frequency of 190/min), for 1 h.The mixtures were then allowed to equilibrate and settle over night atroom temperature. The appearances of the mixtures after one and fivedays a 4° C. were evaluated as: clear colloidal, slightly turbid,turbid, milky, and the results are summarized in Table 5.

TABLE 5 Peptide: Sample LL-37 Galacto- Lipid Appearance after Appearanceafter number (ppm) lipid (%) (w/w) 1 Day 5 Days 01 247 0.135 1:5.5turbid dispersion, turbid dispersion, sediment sediment 02 181 0.1331:7.4 clear colloidal solution clear colloidal solution, slight sediment03 116 0.133 1:11 clear colloidal solution clear colloidal solution 0450.5 0.135 1:27 clear colloidal solution clear colloidal solution 0516.5 0.133 1:81 slightly turbid slightly turbid dispersion, dispersion,homogeneous homogeneous 06 8.2 0.135 1:165 turbid dispersion, turbiddispersion, homogeneous homogeneous 07 — 0.133 — turbid dispersion,turbid dispersion, homogeneous homogeneous 08 248 0.266 1:11 clearcolloidal solution clear colloidal solution, slight sediment 09 1820.267 1:15 clear colloidal solution clear colloidal solution 10 1160.266 1:23 clear colloidal solution clear colloidal solution 11 49.80.268 1:54 slightly turbid slightly turbid dispersion, dispersion,homogeneous homogeneous 12 17.1 0.266 1:156 slightly turbid slightlyturbid dispersion, dispersion, homogeneous homogeneous 13 8.9 0.2651:298 slightly turbid slightly turbid dispersion, dispersion,homogeneous homogeneous 14 — 0.265 — slightly turbid slightly turbiddispersion, dispersion, homogeneous homogeneous 15 247 0.532 1:22 clearcolloidal solution clear colloidal solution 16 182 0.532 1:29 slightlyturbid slightly turbid dispersion, dispersion, homogeneous homogeneous17 116 0.533 1:46 turbid dispersion, turbid dispersion, homogeneoushomogeneous 18 49.2 0.533 1:108 turbid dispersion, turbid dispersion,homogeneous homogeneous 19 16.5 0.534 1:324 turbid dispersion, turbiddispersion, homogeneous homogeneous 20 8.2 0.532 1:649 turbiddispersion, turbid dispersion, homogeneous homogeneous 21 — 0.533 —turbid dispersion, turbid dispersion, homogeneous homogeneous 22 2480.799 1:32 turbid dispersion, turbid dispersion, homogeneous slightsediment 23 182 0.802 1:44 milky dispersion, milky dispersion,homogeneous slight sediment 24 115 0.801 1:70 milky dispersion, milkydispersion, homogeneous slight sediment 25 50.1 0.799 1:159 milkydispersion, milky dispersion, homogeneous slight sediment 26 16.8 0.7991:476 milky dispersion, milky dispersion, homogeneous slight sediment 278.6 0.798 1:928 milky dispersion, milky dispersion, homogeneous slightsediment 28 — 0.798 — milky dispersion, milky dispersion, homogeneousslight sediment

It is clear that certain ratios of LL-37 peptide and galactolipid giverise to an appearance in solution, which indicate the presence of smallcomplexes, smaller in size than particles of the corresponding sampleswithout LL-37. A clear solution indicates a superior colloidalstability.

Example 6 Conformational Measurements

Measurements of circular dichroism (CD) of LL-37 in solution may revealinformation about conformational changes. The antibacterial activity ofLL-37 is dependent on the conformation: a high content of helicalcontent results in a strong antibacterial action and a high cytotoxicactivity (Johansson et al., J Biol Chem 273:3718-3724, 1998). It hasbeen found that the α-helical conformation of LL-37 is dependent on thecounterion, the pH, and the peptide concentration (Johansson et al., JBiol Chem 273:3718-3724, 1998). It is also known that a certain fractionof the peptide has an α-helical structure in aqueous solution and thatthis structure may be promoted by the presence of additives such aslipids, transforming it from a random coil to an α-helix (Turner et al.,Antimicrob Agents Chemother 42:2206-2214, 1998).

Samples for circular dichroism (CD) measurements were prepared in 10 mMaqueous phosphate buffer solution, pH 7.0, containing 200 ppm LL-37 (asthe trifluoroacetate, lot 971/26), with and without 0.40%CPL-Galactolipid. The samples, 20 ml in 50 ml glass flasks, werevigorously shaken with an ST mixer (type B1, E. Büchler, Tübingen) setat 7.5 (corresponding to an approximate frequency of 220/min) for 2 h.They were then allowed to equilibrate and settle over night at 2-8° C.

CD spectra were recorded on a Jasco J-720 (Jasco Inc.)spectropolari-meter. The sample compartment with the cuvette cell (1 mmpath length) was placed near the photomultiplier, in order to reduceeffects of light scattering from the disper-sions. The samples weremeasured at room temperature and scanned from 280 to 200 nm at a rate of20 nm/min, with a resolution of 1 nm and 3 accumulations per run. Theresults are expressed as the mean residue ellipticity, [θ], and thepercentage of α-helical conformation at 222 nm is estimated by thefollowing formula: ([θ]₂₂₂+3900)·100/41900.

The CD measurements on 200 ppm LL-37 in 10 mM phosphate buffer solution,pH 7.0, revealed an α-helical secondary structure by double dichroicminima at 208 and 222 nm. The minimum at 222 nm was used to calculatethe percentage α-helical structure, which was found to be about 63%.When the galactolipid was added at a concentration of 0.40% (w/w) in thesame buffer solution the α-helical structure of LL-37 was practicallyunaffected, with an approximate α-helical structure of 64%.

Enhanced helical conformation is related to increased antibacterialactivity. It is speculated that the secondary structure is also relevantfor the wound healing capacity of LL-37, where a high percentage ofα-helical structure means enhanced activity. In an aqueous buffersolution this also means high cytotoxicity, but in the presence ofgalactolipid the secondary structure is retained, and thus the activityis unaffected, whereas the cytotoxicity is diminished.

An anionic synthetic phospholipid, palmitoyl-oleoyl-phosphatidylglycerol(POPG; Sigma-Aldrich, P6956) was used as a reference and tested usingthe same experimental conditions as described above. A lower percentageof α-helical structure, 58%, was found when this lipid was present,indicating that the conformation and thus activity of LL-37 is moreinfluenced by the negatively charged phospholipid than by the neutralgalactolipid. However, more importantly, after one month of storage at4° C. the sample had partially separated, with sediments on the bottomof the container. Gentle shaking resulted in a coarse dispersion. At thesame time-point, sediments were also observed in the correspondingsample based on galactolipid, but to a lesser extent, which could beredispersed to a fine dispersion by gentle agitation.

Example 7 Cytotoxicity Tests

In vitro cytotoxicity assays are valuable for the evaluation of thetoxicity of materials, which come into close contact with livingtissues.

Selected formulations were tested for in vitro cytotoxicity in culturedmammalian cells (L 929 mouse fibroblasts). The test design was based onthe US Pharmacopeia 26^(th) edition, Method <87> and the ISO 10993-5standard.

Formulations D and E (see Example 4, Table 2) were mixed with completecell culture medium (HAM F12 medium with 10% foetal bovine serum) atconcentrations of 10, 2, 0.4 and 0.08% (v/v). These test solutions wereused to treat triplicate cell cultures for 24 h. Triplicate untreatedcultures, negative controls (treated with an extract of polypropylene)and positive controls (treated with an extract of tin-stabilisedpolyvinyl chloride) were included.

Both formulations showed no to slight toxicity (cytotoxicity grade 0-1)when tested at 10% (v/v) and no toxicity (cytotoxicity grade 0) at 2%,0.4% and 0.08% (v/v).

Cytotoxicity test with a positive control solution containing 100 ppmLL-37 in PBS caused mild toxicity (cytotoxicity grade 2) at all fourconcentrations tested (10, 2, 0.4 and 0.08% mixtures of the solutionwith cell culture medium). This level of toxicity is defined as 20-50%of the cells being dead or showing morphological signs of toxicity. Thescale has a range of 0 to 4 and when test extracts of medical devicesare tested, grades 3 and 4 fail the test. This positive control solutionis considerably more toxic than formulation D and E which showed no orjust slight toxicity.

BIOLOGICAL EXPERIMENTS

Based on our recent findings that

-   -   hCAP18/LL-37 is induced in skin and mucous membranes in        association with inflammation and wounding, and    -   hCAP18/LL-37 is lacking in chronic ulcer epithelium despite        massive inflammation,        we hypothesized that hCAP18/LL-37 is involved in the        regenerative capacity of skin epithelium. The following        experiments were performed to test this hypothesis.

Test 1. Investigation of the Expression Pattern of hCAP18/LL-37 inNon-Inflammatory Human Wound Healing

Tissue Samples

Human skin was obtained from routine abdominal or breast reductionsurgery. Under sterile conditions, full-thickness wounds were made, onthe epidermal side, with a 3-mm biopsy punch. These ex vivo wounds wereexcised with a 6-mm biopsy punch and subsequently transferred to 24-wellplates and covered with 2 ml of medium. Such wounds reproduciblyre-epithelialize within 4-7 days (Kratz et al. Scand J Plast ReconstrSurg Hand Surg 28:107-112, 1994; Inoue et al., J Invest Dermatol104:479-483, 1995; Kratz et al., Microsc Res Tech 42:345-350, 1998).Medium, DMEM (Dulbecco's modified Eagle's medium, GIBCO) containing 10%fetal calf serum (FCS) and antibiotics (PEST=penicillin 50 U/ml andstreptomycin 50 mg/ml), was changed every third day. Wounds wereharvested at different time-points, by 2, 4 and 7 days post-wounding andsnap frozen. In total, the experiment was repeated four times. Fourdifferent donors were used and triplicate wounds were made for eachcondition in every experiment. In each experiment, only skin from asingle donor was used.

Preparation of RNA Probes

To detect mRNA for the hCAP18 gene and immunoreactivity for hCAP18/LL-37we performed in situ hybridization and immunohisto-chemistry on samplesof wounds representing all time-points of sequentialre-epithelialization. For in situ hybridization we used ³⁵S-labeledantisense and sense RNA probes and the experiment was performed asdescribed (Frohm Nilsson et al., Infect Immun 67:2561-2566, 1999).

Preparation of LL-37 Antibody

For immunohistochemistry we raised and prepared a polyclonal LL-37antibody as follows: LL-37 peptide (lot YS 5253, EuroDiagnostica AB,Malmo, Sweden) was prepared as a trifluoroacetate salt according toFmoc-strategy using solid phase synthesis (Fields and Noble, 1990) andpurified by HPLC to a purity of 98%. Biological activity of the peptidewas confirmed in an antibacterial assay. The peptide was used forimmunization of three rabbits according to a standard protocol(AgriSera, Vannas, Sweden). Polyclonal antiserum was affinity-purifiedusing synthetic LL-37 peptide and the purified antiserum was assessedwith ELISA. IgG concentration of the immune serum was diluted to 0.5mg/ml. Pre-immune serum was collected from each rabbit and the IgGconcentration was 2 mg/ml.

Immunohistochemistry

All biopsies were snap frozen and handled identically. In short, 6-7 μmthick cryostat sections were incubated with the LL-37 antibody atdilutions 1:1000 and 1:2000 and stained according to the indirectperoxidase method using a Vectastain kit (Vector Laboratories,Burlingame, USA) and following the manufacturer's instructions. Sectionswere counterstained with Mayer's hematoxyline solution. All experimentswere repeated minimum three times to ensure reproducability. Ascontrols, serial tissue sections were processed in parallel withoutadding primary antibody and using pre-immune rabbit IgG (DAKO, Glostrup,Denmark) as primary antibody.

Results

At time-point 0 h there was moderate expression of hCAP18 mRNA and LL-37protein in the basal layer of the epidermis throughout the tissueconsistent with our previous findings of a constitutive hCAP18expression in basal epidermis. Wounds harvested at different time-pointsduring re-epithelialization demonstrated a distinct signal for hCAP18mRNA and LL-37 protein in the epithelium migrating to cover the woundedsurface. No cells in the underlying dermal matrix were positive forhCAP18/LL-37. These results indicate that de novo synthesis of hCAP18occurs in keratinocytes during re-epithelialization without inflammationand support our hypothesis that hCAP18 may be linked to epithelialregeneration.

Test 2. Inhibition of Re-Epithelializing of Human Skin Wounds Ex Vivowith LL-37 Antibody

LL-37 antibody, prepared in Test 1, was added in 2 ml medium per well(DMEM, +10% FCS and PEST) to a final antibody dilution of 1:10, 1:100and 1:1000. As control we used the corresponding pre-immune serum at afinal IgG concentration equal to the 1:10 dilution of the LL-37antiserum and a set of wounds treated only with medium. Eachexperimental condition was made in triplicates and repeated twice. Themedia were changed every third day and LL-37 antibody or pre-immuneserum was added as described above. The ex vivo wounds were harvested 2,4 and 7 days post-wounding. All specimens were snap frozen, sectioned incompletion and mounted on Superfrost Plus slides prior to staining withhematoxylin-eosine. Sections representing maximal re-epithelializationin the center of the wounds were selected for evaluation. Theproliferative capacity of keratinocytes was investigated throughimmunohisto-chemistry with the proliferation marker Ki67 (mousemonoclonal Ki67 antiserum (DAKO, Glostrup, Denmark) at 1:25 dilution) inwounds representing all treatment conditions.

Results

Treatment with LL-37 antibody produced a concentration-dependantinhibition of re-epithelialization. All wounds treated with the highestLL-37 antibody concentration (1:10) failed to re-epithelialize. In thesewounds only single keratinocytes with a fragile flattened appearance hadmigrated from each wound edge. The wounds treated with LL-37 at mediumconcentration (1:100) showed delayed re-epithelialization, these woundswere mostly healed by day 7 but not by day 4. Moreover, the epitheliumwas thinner and the keratinocytes had a fragile appearance. Woundstreated with LL-37 antibody at the lowest concentration (1:1000) did notdiffer from control wounds, which had all healed by day 4 with a 2-3layer robust epithelium. Control wounds treated with only medium andcontrol IgG antibody healed equally. In the control wounds the majorityof cells in the re-epithelializing tongue were positive for theproliferation marker Ki67, whereas there were no Ki67 positive cells inthe wounds treated with LL-37 at 1:10. We concluded from this experimentthat LL-37 may be critically involved in skin re-epithelialization andthat the proliferative capacity seemed preferentially affected, sinceblocking with LL-37 antibody allowed the initial migration of singlecells from the wound edge, but effectively prevented furtherproliferation of the keratinocytes.

Test 3. Proliferation of HaCat Cells by Treatment with Synthetic,Biologically Active LL-37 Peptide Per Se and in Combination with a PolarLipid Carrier

HaCat cells were used for these experiments. HaCat cells are animmortalized human keratinocyte cell line (Boukamp et al., J Cell Biol106:761-771, 1988), which is suitable for experimental keratinocyteresearch. HaCat cells were cultured in medium (DMEM, +10% FCS and PEST).Both types of cell cultures were treated with synthetic, bioactive LL-37(lot YS 5253). In addition a mixture of LL-37 (114 μg/ml) andCPL-Galactolipid (0.2%) in medium containing serum at either 2 or 10%was added to evaluate the capacity to increase proliferation and inhibitcytotoxicity. Cells were harvested at different time-points (24 h, 48 h,72 h and 96 h) and counted by flowcytometry (Becton-Dickinson) andstained by Trypan-Blue to evaluate viability. Positivity for Trypan-Blueindicates that the cell membrane has been damaged. Proliferation andviability were also ascertained by measuring mitochondrial activity(WST-1, Roche, Cook et al. Anal Biochem 179:1-7, 1989).

TABLE 6 Proliferation of HaCat cells at 96 h Assessed by Flowcytometry.Serum Number Trypan Increased EGF LL-37 Conc. of Cells Blue+Proliferation (nM) (μg/ml) (%) (Mean) (%) (%) — — 10 32270 <1 0 1.7 — 1042000 <1 30 — 25 10 36470 <1 13 — 50 10 40950 <1 27 — 100 10 66430 <1100 — 25 2 32130 <1 0 — 50 2 53620 30-50 Not relevant Cytotoxic effect —100 2 15120 100 Not relevant Cytotoxic effect Increase in cellproliferation is calculated in comparison with baseline (−EGF). Meanvalues from triplicate samples/condition in three separate experimentsare presented

TABLE 7 Proliferation and Viability of HaCat Cells at 48 h Measured byMitochondrial Activity (WST-1). Serum Trypan Increased EGF LL-37 Conc.Blue+ Proliferation (nM) (μg/ml) (%) Absorbance (%) (%) — — 10 0.622 <10 1.7 — 10 1.107 <1 77 — 100 10 1.110 <1 78 Increase in cellproliferation is calculated in comparison with baseline (−EGF). Meanvalues from 6 samples/condition in one experiment are presented

TABLE 8 Proliferation of HaCat Cells at 72 h Assessed by FlowcytometryEGF LL-37 Lipid Serum Number of Trypan Blue + Increased (nM) (μg/ml)(0.2%) Conc. (%) Cells (Mean) (%) Proliferation (%) — — − 10 55207 <1  01.7 — − 10 85050 <1 54 1.7 — + 10 87640 <1 58 — 100 − 10 88853 <1 61 —100 + 10 91980 <1 66 — 100 − 2 150500 100 Not relevant Cytotoxic effect— 100 + 2 87360 <1 58 Increase in proliferation is calculated incomparison with baseline (-EGF). Mean valued from triplicatesamples/condition in one experiment are presented.

Results

The treatment of HaCat cells with LL-37 peptide resulted in aconcentration-dependant increase in proliferation. This indicates thatLL-37 peptide has the capacity to stimulate the proliferation ofkeratinocytes to a level that equals or surpasses that of EGF, thegolden standard for epithelial cell proliferation. We have used EGF at1.7 nM since this has been established as optimal to stimulateproliferation of keratinocytes in culture and has become a standardculture condition (Cohen et al., Dev Biol 12:394-407, 1965). HaCat cellsare highly proliferating epithelial cells and it is interesting thatLL-37 can increase the proliferation of these cells even further. Thecytotoxic effect induced by LL-37 at 100 μg/ml, in 2% serum wascompletely abolished when lipid was added to the mixture, indicatingthat the lipid is able to substitute for serum in this experimentalcondition.

The test has shown that synthetic, bioactive LL-37 (25-100 μg/ml) addedto cell cultures of HaCat cells, in media with 10% Fetal Calf Serum(FCS), increases proliferation in a concentration-dependent manner.However, if the peptide (100 μg/ml) was added to a keratinocyte culturein a medium containing 2% FCS, all of the keratinocytes became positivewith Trypan Blue staining, indicating a cytotoxic effect on these cells.

The cytotoxic activity of cathelicidin is inhibited by the presence ofserum, a mechanism thought to protect the host cells from potentiallyharmful effects. Our data confirms that the cytotoxic effect of LL-37 isinhibited in the presence of serum (10%). In addition, the mixture ofLL-37 (25 μM) and polar lipid carrier (0.2%), in medium containing thelower serum concentration (2% FCS), inhibits the cytotoxic effect andincreases the proliferation. These data suggest that the polar lipidcarrier has similar protecting capacity as serum, without interferingwith the LL-37 bioactivity.

Primary data show that human keratinocytes are proliferated in the sameway as HaCat cells.

Test 4. Proliferation of HaCat Cells by Treatment with the SyntheticPeptides LL-36, LL-37 and LL-38

HaCat cells were cultured in medium (DMEM, +10% FCS and PEST). HaCaTcells were plated in 96 well plates (Falcon, USA) at the concentrationof 2000 cells per well. Cells were plated at −48 hours and stimulatedwith different concen-trations of synthetic LL-37, LL-36, and LL-38peptide by hour 0 and after 48 hours.

The testing was done in one experiment with 6 wells in each condition. 1Ci/mmol of ³H-Thymidine (THYMIDINE, [METHYL-³H]-740.0 GBq/mmol (20.00Ci/mmol) 1.0 ml of Ethanol:Water, 7:3, Perkin Elmer Life Sciences Inc.Boston Mass., USA) was added to each well and incubated for 12-17 hours.Proliferation was evaluated by ³H-Thymidine incorporation a liquidscintilator (MicroBeta Perkin Elmer Life Sciences Inc. Boston Mass.,USA) after 72 and 96 hours.

TABLE 9 Proliferation of HaCat Cells by LL-37 at 96 h Assessed by³H-Thymidine Incorporation after 72 and 96 Hours. Serum Counts PerStandard Increased LL-37 Conc. Minute Deviation Proliferation (μg/ml)(%) (Mean) (+/−) (%) 0 10 52774 11639 0 1.00 10 75445 32827 43 5.00 10102353 33808 94 10.00 10 73548 8424 39 25.00 10 76510 10550 45 50.00 1065119 8565 23 Increase in cell proliferation (Proliferation Index) iscalculated in comparison with baseline (Control = 0 μg/ml). Mean valuesfrom four samples per condition in one experiment are presented.

TABLE 10 HaCat cells stimulated by LL-36 peptide. Proliferation Assessedby 3H-Thymidine Incorporation after 96 Hours. Serum Counts Per StandardIncreased LL-36 Conc. Minute Deviation Proliferation (μg/ml) (%) (Mean)(+/−) (%) 0 10 69323 7511 0 1.00 10 86253 10770 24 5.00 10 116381 1457068 10.00 10 70157 3660 1 25.00 10 72674 7965 5 50.00 10 68560 11699 −1Increase in cell proliferation (Proliferation Index) is calculated incomparison with baseline (Control = 0 μg/ml). Mean values from foursamples per condition in one experiment are presented.

TABLE 11 HaCat Cells Stimulated by LL-38 Peptide. Proliferation Assessedby 3H-Thymidine Incorporation after 96 Hours. Serum Counts Per StandardIncreased LL-38 Conc. Minute Deviation Proliferation (μg/ml) (%) (Mean)(+/−) (%) 0 10 79191 15277 0 1.00 10 82008 7911 4 5.00 10 68694 16599−13 10.00 10 57293 8512 −28 25.00 10 54294 14335 −31 50.00 10 48701 6080−39 Increase in cell proliferation (Proliferation Index) is calculatedin comparison with baseline (Control = 0 μg/ml). Mean values from foursamples per condition in one experiment are presented.

Test 5. Proliferation of Human Fibroblasts by Treatment with LL-37Peptide

The peptide LL-37 used in this and following tests was as described inExample 1 (lot 971/26). The fibroblasts, a type of stromal cells, wereobtained from injured and uninjured skin in patients with chronic legulcers due to venous insufficiency. Punch-biopsies (4-mm) were takenfrom the wound margin including 50% of the epithelialized area and fromuninjured skin in the knee region. Individuals with a history ofdiabetes mellitus, arterial insufficiency or chronic inflammatorydisease were excluded. Patients with signs of eczema in the ulcermargin, clinical signs of infection or undergoing systemic or topicalantibiotic treatment at the time for biopsy were also excluded. Patientsincluded were all treated with inert local dressings and standardcompression bandaging.

Fibroblast were put in culture using explant technique (Hehenberger etal., Cell Biochem Funct 15:197-201, 1997). Fibroblasts were plated in 96well plates (Falcon, USA) at the concentration of 2000 cells per well.Cells were plated at −48 hours and stimulated with differentconcentrations of synthetic LL-37 peptide by hour 0. The testing wasdone in one experiment with 6 wells in each condition. Proliferation andviability were ascertained by measuring mitochondrial activity (WST-1,Roche) after 24 h and 48 h. See Table 12 and Table 13 below. Increase incell proliferation (Proliferation Index) is calculated in comparisonwith baseline (Control=0 μg/ml). Mean values from six samples percondition in one experiment are presented.

TABLE 12 Human Wound Fibroblast Stimulated by LL-37. Proliferation andViability of Human Fibroblasts Measured by Mitochondrial Activity(WST-1) at 48 Hours. Serum Standard Increased LL-37 Lipid Conc.Deviation Proliferation (μg/ml) (0.2%) (%) Absorbance (+/−) (%) — − 100.785 0.020 0 25 − 10 1.171 0.242 49 50 − 10 1.073 0.199 37 100 − 100.955 0.187 22 100 + 2 0.960 0.122 22

TABLE 13 Human Normal Fibroblast Stimulated by LL-37 Peptide.Proliferation and Viability of Human Fibroblasts Measured byMitochondrial Activity (WST-1) at 48 Hours Serum Standard IncreasedLL-37 Conc. Deviation Proliferation (μg/ml) (%) Absorbance (+/−) (%) —10 0.560 0.019 0 25 10 0.597 0.067 7 50 10 0.626 0.076 12 100 10 0.6690.051 19

Test 6. Transgenic Expression of hCAP18 in HEK293 Cells andProliferation of HEK293-hCAP18 Cells

A Bfa1 fragment from Image clone 3057931 (ref) containing the entirecoding sequence for hCAP18 including the 16 by of the 5′-untranslatedregion, was subcloned into the Sma1-site of the bycistronic vectorpIRES2-EGFP (BD Biosciences, Bedford, Mass.). Human embryonic kidneycells, HEK293, were transfected using Fugene (Roche Diagnostics,Indianapolis, Ind.) under standard conditions, and selected for twoweeks with 400 ng/ml G418 antibioticum (Invitrogen, Paisley, UK). Thecells were sorted for EGFP expression with a MoFlo® high speed cellsorting flow cytometer (DakoCytomation, Fort Collins, Colo.) usingSummit™ software for data analysis, and their expression of CAP18 wasquantified by immunoblotting. Control cell lines were similarlyestablished by transfection with the vector only expressing EGFP.

For proliferation assay, cell lines were harvested at 70% confluence andseeded in 24-well plates. After 24 hours, medium was changed and cellswere cultured in 2 ml of medium (OPTIMEM, Gibco BRL, Life Technologies,Scotland) supplemented with 5% FCS and PEST. All conditions wereperformed in triplicates. Medium was changed every second day. Celllines were then harvested at day 6 and counted by Flow Cytometry. Cellviability was measured with Trypan Blue; under all conditions <5% of thecells were Trypan Blue positive. Increase in cell proliferation(Proliferation Index) is calculated in comparison with baseline(HEK293-EGFP). Mean values from triplicate samples per condition in oneexperiment are presented.

TABLE 14 Proliferation of HEK293-hCAP-18 Cells at 144 Hours Assessed byFlow-Cytometry. Serum Standard Increased Cell Conc. Number of DeviationProliferation Type (%) Cells (Mean) (+/−) (%) HEK293-EGFP 5 169063 637260 HEK293-hCAP18 5 485884 88168 187

The proliferation of the HEK293-hCAP18 cells was also assessed byincorporation of ³H-thymidine and the results obtained are presented inTable 15 below. The increase in cell proliferation (Proliferation Index)is calculated in comparison with baseline (HEK293-EGFP). Mean valuesfrom four samples per condition in one experiment are presented.

TABLE 15 Proliferation of HEK293-hCAP-18 Cells at 144 Hours Assessed by³H-Thymidine Incorporation after 96 Hours. Serum Counts Per StandardIncreased Cell Conc. Minute Deviation Proliferation Type (%) (Mean)(+/−) (%) HEK293-EGFP 0.1 364 118 0 HEK293-hCAP18 0.1 796 206 111HEK293-EGFP 0.5 811 459 0 HEK293-hCAP18 0.5 2271 792 180 HEK293-EGFP 1744 433 0 HEK293-hCAP18 1 2303 359 209 HEK293-EGFP 2 767 334 0HEK293-hCAP18 2 3483 771 354 HEK293-EGFP 5 958 414 0 HEK293-hCAP18 56088 1783 534 HEK293-EGFP 10 1806 664 0 HEK293-hCAP18 10 6541 2827 262

Test 7. Culturing of Human Cells for Transplantation in Different GrowthMedia

Culture of Epithelial Cells

A piece of skin, 1×1 cm, is excised from healthy skin of the patient.The skin is minced and treated with trypsin/EDTA (0.05/0.01%) and2-5×10⁶ of the recruited keratinocytes are added to 1.5×10⁶mitomycin-pretreated (4 μg/ml, 2 h) 3T3 cells in 75 cm² culture flasks.Growth medium A containing LL-37 peptide is added. Cells are harvestedby trypsination as sheets and transplanted onto the patient.

Growth Medium A is used for culture of epithelial cells such as e.g.keratinocytes in vitro and consists of Basal Medium and a growthpromoting kit (GPK) including a) LL-37 peptide in a salt solution, b)penicillin+streptomycin, c) insulin, d) transferring, e)triiodotyronine, f) hydrocortisone, g) choleratoxin, and a selectedcytoxicity reducing agent, such as serum or a polar lipid.

Culture of Stromal Cells

Stromal cells are obtained from a 4 mm skin biopsy, cleaned fromsubcutaneous tissue and plated in cell culture dishes using the explanttechnique to obtain primary fibroblasts. Growth medium B is used forculturing the biopsy. Cells are harvested by trypsination and given backto the patient.

Growth Medium B is used for culture of stromal cells such as e.g.fibroblasts in vitro and consists of Basal medium and a growth promotingkit including a) LL-37 peptide in a salt solution, b)penicillin+streptomycin, and a selected cytotoxicity reducing agent,such as serum or a polar lipid.

Basal medium is based on double-distilled water containing inorganicsalts, phenol red, glucose, thymidine, hypoxanthinine, HEPES, sodiumpyruvate, aminopterin, amino acids and vitamins

SUMMARY OF THE EXPERIMENTS

In summary, it has been demonstrated that LL-37 is produced in skinepithelium during normal wound healing and that LL-37 is required forre-epithelializa-tion to occur. We have also shown that endogenous LL-37is lacking in chronic ulcer epithelium. We therefore propose thattreatment with LL-37, as well as with N-terminal fragments of saidpeptide, and functional derivates thereof provides a rational strategyto promote healing of such ulcers. Furthermore, addition of LL-37 andtransgenic expression of hCAP18/LL-37 also stimulates proliferation ofhealthy cells indicating that LL-37 can be used to enhance both normaland deficient epithelial repair in vivo and proliferation of epithelialcells in vitro for autologous cell transplantation. We have alsoidentified a suitable carrier and delivery system that reducescytotoxicity and has the potential to protect from rapid degradation invivo of LL-37 and other cathelicidin peptides.

The invention claimed is:
 1. A method for stimulating proliferation ofepithelial cells in a chronic ulcer due to diabetes, comprisingadministering an effective amount of a peptide LL-37 (SEQ ID NO: 1) or apharmaceutically acceptable salt thereof to a subject in need of suchtreatment.
 2. The method according to claim 1, wherein said peptideLL-37 (SEQ ID NO: 1) is in the form of the acetate salt.
 3. The methodof claim 1 wherein the peptide LL-37 (SEQ ID NO: 1) is administeredtopically to the chronic ulcer.
 4. A method for stimulating epithelialregeneration in a chronic ulcer due to diabetes, comprisingadministering an effective amount of a peptide LL-37 (SEQ ID NO: 1) or apharmaceutically acceptable salt thereof to a subject in need of suchtreatment.
 5. The method according to claim 4, wherein said peptideLL-37 (SEQ ID NO: 1) is in the form of the acetate salt.
 6. The methodof claim 4 wherein the peptide LL-37 (SEQ ID NO: 1) is administeredtopically to the chronic ulcer.
 7. A method for stimulatingre-epithelialization in a chronic ulcer due to diabetes, comprisingadministering an effective amount of a peptide LL-37 (SEQ ID NO: 1) or apharmaceutically acceptable salt thereof to a subject in need of suchtreatment.
 8. The method according to claim 7, wherein said peptideLL-37 (SEQ ID NO: 1) is in the form of the acetate salt.
 9. The methodof claim 7 wherein the peptide LL-37 (SEQ ID NO: 1) is administeredtopically to the chronic ulcer.